Dengue (DEN; Flaviviridae) viruses continue to pose health problems for human populations in tropical regions of the world. DEN hemorrhagic fever and DEN shock syndrome are threatening large proportions of the urban and rural populations in Asia and the Americas. Concomitant with the spread of disease is the rapid proliferation of the DEN virus mosquito vector, Aedes aegypti. The lack of effective vaccines against DEN, as well as the emergence of pesticide resistant vectors, has had a major negative impact on developing conventional strategies for controlling the disease. The long term goal of this research is to develop molecular strategies that reduce or eliminate virus replication in the vector and prevent virus transmission. We have shown that expression of sense or antisense RNA of the DEN prM gene from a Sindbis (SIN; Togaviridae) virus vector in mosquito cells or adult Ae. Aegypti effectively interferes with replication of superinfecting DEN virus. In this proposal, we will use SIN transducing viruses to express second generation anti- DEN virus agents in Aedes aegypti. These agents will include: 1)sense and antisense RNAs of improved design that can be targeted to all 4 serotypes of DEN virus, 2) mutated DEN replicase gene NS5, and 3) anti-DEN ribozymes. Rationally designed, DEN sense and antisense RNAs should complement and potentially enhance RNA mediated resistance strategies that have already been shown to be effective. Initially, SIN transducing viruses will be used to express a model ribozyme targeted to trans-cleave a Beta- galactosidase gene expressed in transformed mosquito cells. The utilization of ribozyme technology to reduce or ablate the competency of mosquitoes for DEN viruses is a natural extension of the antisense strategies already in use. The expression in mosquitoes of mutated forms of the DEN polymerase (NS5) gene represents important strategies for ablating virus replication in the vector and its efficacy as an antiviral agent needs to be assessed. Additionally, promising second-generation anti virus agents will be analyzed in transformed mosquito cell culture to study the efficacy of these agents when delivered from a DNA expression system. Finally, promising interference strategies developed from SIN virus transduction and DNA expression of the anti-virus agents will be tested in vivo using Aedes densonucleosis (AeDNV; Parovoviridae) virus. In toto, this work should give us significant understanding of which anti-DEN virus strategies are important for ultimately generating transgenic mosquitoes that are resistant to DEN virus infections.